Osteoblast Stimulating Orthopedic Implant

ABSTRACT

Nanomaterials for neural and orthopedic prostheses are disclosed. Composite carbon nanofibers enhance neuronal growth and minimize glial scar tissue formation. Methods and compositions to promote neuronal growth and minimize scar tissue formation during prolonged monitoring and treatment of neural tissue are disclosed. Composite polyurethane carbon nanofiber is a suitable material for neural implant. Composite carbon nanomaterials decrease adhesion of astrocytes and fibroblasts

This application claims priority to U.S. Ser. No. 60/458,199 filed 27Mar., 2003.

FIELD

This disclosure generally relates to implantable neural and orthopedicdevices having therein or thereon a nanomaterial.

BACKGROUND

Many orthopedic implants are made of vanadium steel, stainless steel,cobalt alloys, titanium, gold and amalgams (a metal alloy containingmercury). For neural implants, silicon is most widely used.

A healing response that follows an implantation, impacts the long-termfunctionality of the implanted device. The prolonged healing processrecruits a variety of body fluids, proteins, and unwanted cell types tothe tissue-implant interface. Healing characteristics common tounsuccessful bone and neural prosthetic wound-healing include prolongedfibrous encapsulation and chronic inflammation.

Extensive scar tissue formation is a common occurrence that leads toimplant failure for an orthopedic implant juxtaposed to bone or a neuralimplant with the conductive neural tissue. With regard to orthopedicimplants, fibrous encapsulation (or callus formation) at thetissue-implant interface decreases the effectiveness of the implant'sbonding, to bone and often results in clinical failures. With regard toneural implants, glial scar-tissue forms around probes implanted intothe central nervous system. The glial scar response, gliosis, includesthe formation of cells, for example astrocytes, which encapsulate theprobes with non conductive tissue. Gliosis inhibits the ability of theprobes or any other implants to properly interact with the surroundingneural tissue.

SUMMARY

Methods and compositions that comprise a nanomaterial as a suitablebiocompatible material for neural and orthopedic implants are disclosed.Methods to minimize glial scar tissue formation or fibroblastencapsulation due to prolonged use of implants are disclosed. The carbonnanofibers in a neural or an orthopedic implant range in size of about 2to 200 nm. The nanomaterial can comprise a composite carbonnanofiber-polyurethane matrix. The matrix may be selected from the groupconsisting of polyurethane, polymethacrylate, polyester, polyvinyl andany copolymers thereof. The nanomaterial may be applied to or made apart of the surface of the implant.

More specifically with regard to a neural implant, the implant can be adevice coated with a nanomaterial. The neural implant can be a devicewith at least one component of the device made of a nanomaterial. Theneural implant can comprise carbon nanofibers. The neural implant canhave the carbon nanotubes functionalized. The neural implant can havethe carbon nanotubes aligned.

The neural implant may be a neural probe. The neural implant may be aprostheses that includes an implantable device with a compositepolyurethane carbon nanotube, the device capable of stimulating neuronalgrowth and minimizing glial scar tissue formation. The neural prostheseshas carbon nanotubes which form 2% to 100% of the composite.

One suitable method for minimizing scar formation caused by a neuralimplant includes obtaining a neural implantable device; coating theimplantable device with a nanomaterial; and securing the implantabledevice in the neural tissue.

One suitable method of stimulating neuronal growth and minimizing scarformation by an implant in a brain can comprise obtaining a neuralimplantable device comprising a nanomaterial; securing the implantabledevice in the brain; and providing neuronal stimulants through thedevice.

The orthopedic prostheses can comprise an implantable device coated witha composite polyurethane carbon nanotube, capable of stimulatingosteoblast proliferation and minimizing fibroblast encapsulation.

One suitable method of stimulating osteoblast proliferation andminimizing fibroblast encapsulation by an orthopedic implant, comprisesobtaining an orthopedic implantable device comprising a nanomaterial;and securing the implantable device.

One suitable method of selecting a nanomaterial suitable for implant,comprises determining structural dimensions of a biological molecule ina biological tissue; and fabricating the nanomaterial whose surfacestructural dimension is similar to the biological molecule.

A method of selecting a nanomaterial, wherein the biological molecule islaminin, is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are provided to illustrate some of the embodiments of thedisclosure. It is envisioned that alternate configurations of theembodiments of the present disclosure may be adopted without deviatingfrom the disclosure as illustrated in these drawings.

FIG. 1 illustrates the representative Scanning Electron Microscope (SEM)images of polyurethane: carbon nanotube (PU:CN) composites. Scale bar=1μm. (Sigma), and counted using fluorescence microscopy (365 nmexcitation and 400 nm emission wavelengths).

FIG. 2 shows an optimal neurite extension for neurons cultured on allPU:CN materials. Time=3 d. Values are mean+/−SEM; n=3; p<0.1 compared to100:0 PU:CN wt %. matrix.

FIG. 3 illustrates the representative images of optimal neuriteextension for neurons cultured on all PU:CN materials. Scale bar=1.0 μm;scale bar=20 μm.

FIG. 4 shows decreased astrocyte cell adhesion density with increasednumbers of CNs in PU. Values are mean+/−SEM; 17=3; p<0.1 compared to100:0 PU:CN wt %.

FIG. 5 shows increased osteoblast cell adhesion density with increasednumbers of CNs in PU. Values are mean+/−SEM; n=3; p<0.1 compared to100:0 PU:CN wt %.

FIG. 6 shows decreased fibroblast cell adhesion density with increasednumbers of CNs in PU. Values are mean+/−SEM; n=3; p<0.1 compared to100:0 PU:CN wt %.

FIG. 7 shows high magnification scanning electron micrographs of thefollowing carbon fiber discs: (a) conventional fiber with low surfaceenergy, (b) conventional fiber with high surface energy, (c) nanophasefiber with low surface energy, and (d) nanophase fiber with high surfaceenergy. Original magnification=10,000×; 5 kV; bar=1 micron.

FIG. 8 shows a high magnification scanning electron micrographs ofvarying compositions (by weight) of polycarbonate urethane (PCU) and 60nm carbon nanophase fibers (CN) with high surface energy: (a) 100:0(PCU:CN), (b) 98:2, (c) 90:10, and (d) 75:25. Original magnification=(a)15,000×; (b)-(d) 10,000×; 5 kV; bar=1 micron.

FIG. 9 shows astrocyte adhesion on low and high energy surfaces:borosilicate glass (reference substrate), conventional fibers (200 nm)with low surface energy (SE), conventional fibers (125 nm) with highsurface energy, nanophase fibers (100 nm) with low surface energy, andnanophase fibers (60 nm) with high surface energy; all under standardcell culture conditions for 1 hour. Values are mean±SEM; n=3; * p<0.1(compared to respective nanophase fiber with similar surface energy).

FIG. 10 shows astrocyte adhesion cultured on the following substrates:borosilicate glass (reference substrate), compositions (by weight) ofpolycarbonate urethane (PCU) and carbon nanophase fiber (CN; 60 nm withhigh surface energy) PCU:CN substrates: 100:0, 98:2, 90:10, 75:25, and0:100; all under standard cell culture conditions for 1 hour. Values aremean±SEM; n=3; * p<0.1 (compared to 100:0 PCU:CN wt. %).

FIG. 11 shows astrocyte proliferation cultured on the followingsubstrates: borosilicate glass (reference substrate), conventionalfibers (200 nm) with low surface energy (SE), conventional fibers (125nm) with high surface energy, nanophase fibers (100 nm) with low surfaceenergy, and nanophase fibers (60 nm) with high surface energy.Astrocytes were cultured under standard cell culture conditions for 1,3, and 5 days. Values are mean±SEM; n=3; * p<0.1, ** p<0.05, * * *p<0.01 (compared to respective nanophase fiber with similar surfaceenergy).

FIG. 12 shows normalized astrocyte alkaline phosphatase activitycultured on the following substrates: borosilicate glass (referencesubstrate), conventional fibers (200 mm) with low surface energy (SE),conventional fibers (125 nm) with high surface energy, nanophase fibers(100 nm) with low surface energy, and nanophase fibers (60 nm) with highsurface energy. Intracellular alkaline phosphatase activity (Sigmaunits/mg protein/square cm) was determined after 7 and 14 days. Valuesare mean±SEM; n=3; * p<0.13, ** p<0.08 (compared to low surface energynanophase fiber).

FIG. 13 shows an alignment of carbon nanofibers for neural applications.Arrow indicates alignment.

DETAILED DESCRIPTION OF THE DISCLOSURE

While the concepts of the present disclosure are illustrated anddescribed in detail in the drawings and the description below, such anillustration and description is to be considered as exemplary and notrestrictive in character, it being understood that only the illustrativeembodiment is shown and described and that all changes and modificationsthat come within the spirit of the disclosure are desired to beprotected.

As used herein nanotubes, nanoparticles, nanofibers, nanowires, andnanophase material refer to nanomaterials whose size range is from 1 nmto less than 1 μM. In other words, the nanomaterials have a size rangein nanometers (10⁻⁹ m). As used herein, the term nanomaterial may alsoinclude a nanocomposite that includes a polymer matrix. For example, acarbon nanofiber-polyurethane (CN:PU) or a polyurethane-carbon nanofiber(PU:CN) is a nanocomposite. The term “nanocomposite” generally refers toa composite polymer material comprising at least a plurality ofnanoparticles disposed in any suitable manner on the composite material.The composite may also be referred as a matrix or a polymer substrate orgenerally a substrate. The carbon nanofibers as used herein refer tosingle walled nanotubes (SWNT), double wall nanotubes (DWNT) ormultiwalled nanotubes.

Neural and orthopedic prostheses or implants include devices and probesfor monitoring, catheters and implants for drug delivery, loops, guidewires, valves, artificial vessels, neural bridges, shunts, and stentsfor cell repair and maintenance, and other prostheses that are used forcontinuous monitoring and treatment of affected tissue. An exemplarysilicon electrode probe for neural applications is disclosed in Hetke etal., (2003), the disclosure of which is incorporated herein by reference(Hetke, J. F. and Anderson, D. J., “Silicon electrodes for extracellularrecording,” In: Handbook of Neuroprosthetic Methods, (Finn and LoPresti,eds.), CRC Press LLC, pp. 163-191, 2003). Fabrication methods for probesused in neural applications is described by Najafi, K (1997) in Najafi,K. “Micromachined systems for neurophysiological applications.”,Handbook of microlithography, micromachining, and microfabrication,Volume II: Micromachining and Microfabrication, SPIE, 1997, thedisclosure of which is hereby incorporated by reference. Otherpublications that discuss fabrication methodologies for neuralprostheses include Neuman et al., Fabricating Biomedical Sensors withThin-Film Technology, IEEE EMB Mag. 13: 409-419, 1994 and Wise et al.,Microfabrication Techniques for Integrated Sensors and Microsystems.Science 254: 1335-1342, 1991.

U.S. Pat. No. 5,698,175 relates to a process for purifying, uncappingand chemically modifying carbon nanotubes, and more particularly to aprocess for easily obtaining highly purified nanotubes for use in thefield of chemistry, drugs and electronics, the disclosure of which ishereby incorporated by reference.

U.S. Pat. No. 6,063,243 relates generally to nanotube fabrication andmore specifically to manufacture of nanotubes containing boron, carbonand nitrogen, the disclosure of which is hereby incorporated byreference. U.S. Pat. No. 5,365,073 relates to a nanofabricatedelectroconductive structure and a method of fabrication thereof using aprobe for use in a scanning tunneling microscope (STM), the disclosureof which is hereby incorporated by reference.

There are structural similarities between the nanomaterials and thecomponents of some of the biological organs, particularly the bone andthe neural system at the topographical level. For example, livingsystems are governed by molecular behavior at nanometer scales. For boththe orthopedic and neural implants, surface properties determine theinteract of the implants with the surrounding cells that are in contactwith the biomaterial surfaces after implantation. Topography ofbiological molecules such as proteins, in tissue types, is a factor indesigning suitable nanomaterials.

The molecular building blocks of life—proteins, nucleic acids, lipids,carbohydrates, proteins, and the like—are examples of materials thatpossess unique properties determined by their size, folding, andpatterns at the nanoscale. For example, with respect to bone,hydroxyapatite (the major inorganic component of bone) is between 2 and5 nm in width and 50 nm in length, while type I collagen (the majororganic component of bone) is a triple helix 300 nm in length and 0.5 nmin width, and has a periodicity of 67 nm. For neural tissue, laminin isa key extracellular matrix component that has a cruciform configurationof approximately 70 nm in width and length. The structural dimensions ofbiological molecules is in contrast to the surfaces provided bytraditional implants which have constituent micron grain or particlessizes that provide topographies that do not resemble biologicalsurfaces.

Silicon, a standard material that has been used as neural probes tendsto induce significant glial scar tissue formation. This gliotic responseis mediated largely by astrocytes and forms at implant and injury sites.Gliotic scar tissue is a common difficulty in the field of neuralprosthetics and cause impairment of implant functionality, especiallywith chronic implant applications. The formation of gliotic scar tissueresults in increased electrode impedance around the implant, decreasedlocal density of neurons, and reduced axonal regeneration.

Physiological surfaces such as extracellular matrices that cellsnormally interact with are composed of proteins with nanoscale surfacedimensions. We designed synthetic materials using nanofibers to mimicthe properties of natural tissues as part of a method to minimizereactions such as, for example, the foreign body response and scartissue formation.

In an embodiment, an exemplary neural or orthopedic implant includes apolyurethane (PU)-carbon nanofiber (CN) composite forming a part of orover its surface. The composite has the nanomaterial in the range ofabout 2 to 500 nm, wherein the carbon nanofibers is in a range of about2% to 100% by weight and the composite polyurethane is in a range ofabout 98% to 0% by weight.

In an embodiment, an exemplary neural or orthopedic implant includes ananomaterial forming a part of or over the implant's surface, whereinthe nanomaterial is in the range of about 2 to 200 nm.

In an embodiment, an exemplary neural or orthopedic implant includes ananomaterial forming a part of or over the implant's surface, whereinthe nanomaterial is in the range of about 10 to 100 nm.

In an embodiment, the carbon nanofibers are aligned in a particularorientation to promote axonal or neuronal growth. In an embodiment, thecarbon nanofibers are functionalized, for example, with an agent such as4-hydroxynonenal.

In an embodiment, an exemplary neural or orthopedic implant includes apolyurethane (PU)-carbon nanofiber (CN) composite forming a part of orover the implant's surface. The composite has the nanomaterial in therange of about 10 to 100 nm and with a load density of about 80:20 (wt %CN:PU).

In an embodiment, an exemplary neural or orthopedic implant includes apolyurethane (PU)-carbon nanofiber (CN) composite forming a part of orover the implant's surface, wherein the composite has a nanomaterial inthe range of about 10 to 100 nm and with a load density of about 90:10(wt % CN:PU).

In an embodiment, an exemplary neural or orthopedic implant includes apolyurethane (PU)-carbon nanofiber (CN) composite forming a part of orover the implant's surface, wherein the composite has a nanomaterial inthe range of about 10 to 100 nm and with a load density of about 90:10(wt % CN:PU).

In an embodiment, an exemplary neural or orthopedic implant includes apolyurethane (PU)-carbon nanofiber (CN) composite forming a part of orover the implant's surface, wherein the composite comprisessubstantially of a nanomaterial in the range of about 10 to 100 nm.

The nanomaterial can be coated over the entire device or to a portion ofthe device by dipcoating, spraying, molding, or by any other suitablemethod. The device can also be formed from the nanomaterial. Thecomposite nanomaterial can also be layered or glued on the device.

A neural implant coated with or made from a suitable nanomaterial of thepresent disclosure would be secured in the neurological tissue usingknown techniques to minimize scar formation. The nanomaterial in theimplant may also be aligned to guide axonal growth if necessary. Thenanomaterial may also be functionalized to promote better bonding withthe surrounding tissue. The implant may also provide neuronal stimulantssuch as, for example, nerve growth factors, to promote neuronal growth.A neural implant can be a neural probe for prolonged monitoring,diagnosis, and treatment of neurological disorders.

An orthopedic implant coated with or made from a suitable nanomaterialof the present disclosure would be secured in a desired tissue usingknown techniques to minimize fibroblast encapsulation.

The composite nanomaterials were constructed using carbon nanofibers anda polycarbonate urethane matrix (PU or PCU). The carbon fibers used,were selected from multiwalled carbon fibers with four differentdiameters (from 60 to 200 nm). They were synthesized using catalytic andchemical vapor deposition methods and were acquired from AppliedSciences, Inc./Pyrograf Products, Inc. (Cedarville, Ohio). The fiberswere separated into two groups—conventional (with diameters greater than1.00 nm, specifically 125 and 200 nm), and nanophase (with diameters of100 nm or less, specifically 60 and 100 nm). A high surface energy(125-140 mJ/m²) and low surface energy (25-50 mJ/m²) fiber wasrepresented in each group. The low surface energy fiber was left asgrown, and the high surface energy fiber was obtained by pyrolyticstripping of the carbon fiber to remove the outer hydrocarbon layer.Each type of carbon fiber was uniaxially pressed using a steel-tool dieat 4000 psi for 3 minutes at room temperature to obtain a disc (1.327cm² surface area) for cytocompatibility studies. The discs were thenexposed to ultraviolet radiation for sterilization for 15 min.

The PU used was an FDA-approved polycarbonate urethane (PU orCarbothane, catalogue no PC-3575A, Thermedics) matrix with a specificweight of 1.15 g cm⁻³ was used as the substrate. PU was chosen becausePU is a thermoelastic elastomer, with good mechanical (ultimateelongation 470%) and oxidative stability properties, but isnon-conductive as described in Thapa et al., (2003).

Other matrix or polymer composite materials include, for example,polymers such as polyurethanes, polyvinyls, polyesters, polyacyrlates,polymethacrylates, copolymers thereof or any other thermostable,biocompatible matrix material that interacts compatibly with thenanoparticles and the surrounding tissue.

Composites were formed from carbon fibers and polycarbonate urethane(PU) matrix in different proportions. Compositions with variedpolycarbonate urethane (Thermedics Polymer Products; PC3575A) to carbonnanofiber (CN) weight percents were used (PU:CN—100:0, 98:2, 90:10,75:25). Polycarbonate pellets were allowed to dissolve in chloroform for1 hour, while the carbon nanofibers were sonicated in chloroform for 1hour, and then the two solutions were mixed. This mixture was thensonicated for 1 hour, poured into Teflon petri dishes, and cured in avacuum overnight. Discs (with a surface area of 1.327 cm²) were cut fromthe polymer for cell adhesion experiments. All material sample (discs)(except the monolithic control CNs with 0 and 100% CN by weight) werealso separately degreased and sonicated in acetone and ethanol for 10min according to Thapa et al., (2003). All samples (discs) were exposedto ultraviolet light for 15 min on each side for sterilization purposesprior to experiments with cells. Borosilicate glass coverslips (FisherScientific) were used as reference substrates. The coverslips weredegreased and sonicated sequentially with acetone and ethanol and werethen etched with 1 N NaOH. Autoclaving was used for sterilization.

The nanomaterials and or the matrix materials can be functionalized toenhance interaction between the matrix material and also the biologicalsurroundings. Functionalization of nanomaterials can be achieved withorganic and inorganic agents. One such functionalization agent is4-hydroxynonenal.

The composite materials' effectiveness for reducing the glial scarresponse was tested by way of an adhesion, proliferation, phosphataseactivity, and protein content assays with astrocytes. These assays aredescribed in detail in the Materials and Methods section of thisdisclosure.

Adhesion of astrocytes and proliferation on various compositenanomaterials were decreased in vitro. Decreased adhesion of astrocytesleads to reduced glial scar tissue formation in vivo. Astrocyte adhesionwas decreased with increasing numbers of CNs in a PU matrix (FIG. 4).Although the number of astrocytes was statistically similar between100:0 and 98:2 PU:CN wt %, significantly (p<0.1) fewer astrocytesadhered on 90:10, 75:25, and 0:100 PU:CN wt % compared to 100:0 PU:CN wt%. The smallest number of astrocytes adhered on 75:25 and 0:100 PU:CN wt% formulations; about half as many astrocytes adhered on theseformulations as on 100:0 PU:CN wt %. Since prolonged extensive functionsof astrocytes lead to unwanted glial scar-tissue formation, these datademonstrate that increased numbers of CNs in a PU matrix minimize glialscar-tissue formation that limits neural implant device functionality.Cell culture data strongly indicates that some or all parts (bothnon-conductive and conductive) of a neural prosthetic device could befabricated by altering the amount of CN content in PU while stillmaintaining the ability to limit astrocyte function and promote neuronfunction. An optimal balance of CN:PU may be achieved such that bothneuron extension and decreased astrocyte adhesion is enabled. Forexample, a CN:PU ratio of 90:10% is an embodiment.

Astrocytes (glial scar tissue-forming cells) were seeded onto varioussubstrates for adhesion, proliferation, and long-term function studies(such as total intracellular protein and alkaline phosphatase activity).Results indicated that astrocytes preferentially adhered andproliferated on carbon fibers that had the largest diameter and thelowest surface energy. Composite substrates were formed using differentweight percentages (0-25 wt. %) of the nanophase, high surface energyfibers in a polycarbonate urethane matrix. Results indicated decreasedadhesion of astrocytes with increasing weight percents of the highsurface energy carbon nanofibers in the polymer composite; this furtherdemonstrates that formulations containing carbon fibers in the nanometerregime limit astrocyte functions leading to decreased glial scar tissueformation. Positive interactions with neurons, and, at the same time,limited astrocyte functions leading to decreased gliotic scar tissueformation are required for increased neuronal implant efficacy.

Scanning electron micrographs at high magnification provided evidence ofthe varying fiber diameters (FIG. 7). The scanning electron micrographsfor the composites of polycarbonate urethane and 60 nm carbon fibersrevealed increasing fiber composition per increasing weight percent ofcarbon (FIG. 8). The fibers caused an increased surface roughness in the75:25 weight percent (PCU:CN) composite. Electron spectroscopy forchemical analyses confirmed that the disc surfaces were composedprimarily of carbon (TABLE 3). The results indicated the presence ofsmall amounts of oxygen on the discs, although less was found on thehigh surface energy (125-140 mJ/m²) fiber discs than on the low surfaceenergy (25-50 mJ/m²) discs (2.5+0.6 to 3.0+0.2% compared to 1.0+0.2 to1.7+0.5%). The high surface energy conventional and nanophase carbonfiber discs did show a slight sulfur contamination of 0.4+0.1 to0.5+0.1%, respectively.

The resistivity of the composites decreased exponentially as the weightpercent of carbon nanofibers in PCU composites increased (TABLE 2).These values ranged from 20,500 Ωm for the 98:2 (PCU:CN wt. %) compositeto 0.354 Ωm for the 75:25 composite. This wide range of values indicatesgreat flexibility in design of electrical properties for these materialsby just varying the weight percentage of carbon nanofibers.

After one hour, astrocytes had preferentially adhered to the low surfaceenergy, conventionally sized carbon fiber disc (FIG. 10). This resultwas significantly (p<0.1) greater at 123% higher cell density thanadhesion to the nanophase fiber with similar surface energy. The numberof astrocytes that adhered to high surface energy fibers of both thenanophase and conventional size regimes was similar.

Adhesion to the pure polycarbonate urethane was greater than to most ofthe composites containing the 60 nm carbon fiber (FIG. 10). In otherwords, substrate compositions of 90:10 and 75:25 (PCU:CN by wt. %) hadsignificantly less (p<0.1) adherent cells when compared to polycarbonateurethane (100:0). The differences in adhesion between the 98:2, 90:10,75:25, and 0:100 composites were not statistically significant.

At the 1, 3, and 5 day time points, the cell density was greater on thelow surface energy conventional carbon fiber disc, than on the lowsurface energy nanophase substrate (FIG. 11). This result wassignificant at each time point (p<0.1 at one day, p<0.05 at 3 days, andp<0.01 at 5 days), and was up to 66% greater cell density at 5 days.Compared to the nanophase high surface energy fiber, the conventionalhigh surface energy fiber had significantly more cells after 3 days(p<0.05); more cells were on the conventional high surface energy fiberafter 5 days, but this was not statistically different from nanophasehigh surface energy fibers.

The alkaline phosphatase activity assay was normalized by dividingrespective values of the total intracellular protein content and thesubstrate surface area. At 7 days, there was not a significantdifference in alkaline phosphatase production between the astrocytecells cultured on the four different carbon fibers (FIG. 12). Alkalinephosphatase production was significantly reduced at 14 days on the lowsurface energy, nanophase fiber when compared to the other three typesof fibers (p<0.13 for the conventional high surface energy fiber; andp<0.08 for the conventional low surface energy and nanophase highsurface energy fibers). Compared to all other carbon fibers, this resultrevealed 70 to 93% less of this enzyme produced on the low surfaceenergy carbon nanofiber.

Astrocytes exhibited significantly increased cell density on low surfaceenergy and conventionally sized (with a diameter greater than 100 nm)carbon fibers at 1 hour and 1, 3, and 5 day time periods. Nanofibersminimized astrocyte interactions. Alkaline phosphatase productionindicated that nanophase fibers reduced astrocytic activity even afterprolonged time periods of 14 days.

A 60 nm high surface energy carbon nanofiber was used for furtherinvestigation. Matrix modifications of carbon nanofibers enhances bulkmechanical and electrical properties. Polycarbonate urethane (PCU) wasused as an exemplary matrix polymer based on its proven. clinicalapplications. The carbon nanofibers were combined with polycarbonateurethane to obtain nanoscale carbon fibers in a polymer matrix havingdesirable mechanical properties. The compositions tested (90:10, 75:25;PCU:CN wt. %) had significantly less adhesion of astrocytes than thepure polymer. Nanophase carbon fibers decreased astrocyte adhesion in apolymer matrix by creating a surface with a high degree of biologicallyinspired nanometer roughness. Furthermore, the 75:25 composition hadsimilar adhesion to the pure carbon nanofiber disc, and the electricalresistivity of this composite was also similar to the pure carbonnanofiber disc (0.354 compared to 0.0598 Ωm, respectively). Thus, thebenefits of using PCU with carbon nanofibers is realized with theflexibility of a range of electrical properties (such as conductivity)available for designing materials important for neural applications.Astrocyte function was minimized on carbon nanofibers.

Interactions of osteoblasts was enhanced and adhesion of fibroblasts wasdecreased with nanomaterials. Adhesion of osteoblasts increased withincreasing weight fractions of CNs in PU (FIG. 5). With only a 2 wt %increase of CNs in the PU matrix, significantly (p<0.1) more osteoblastsadhered. Up to three and four times the number of osteoblasts thatadhered on 100:0 PU:CN wt % adhered on 90:10 and 75:25 PU:CN wt %composites, respectively. Statistically similar numbers of osteoblastsadhered on 75:25 compared to 0:100 PU:CN wt % formulations.

Adhesion of fibroblasts was decreased on PU composites with increasingnumbers of CNs (FIG. 6). Similar numbers of fibroblasts were measured on100:0 and 98:2 PU:CN wt %; however, the number of adherent fibroblastssignificantly decreased with 10 and 25 wt % of CNs compared to 100 wt %PU. Prolonged functions of fibroblasts at the implant interface limitsnecessary osseointegration events and results in callus formation thatmay lead to implant loosening and eventual failure. Materials (such asthe CNs in a PU matrix that this study demonstrated) which increasefunctions of osteoblasts while at the same time decreasing functions offibroblasts have the potential to effectively bond to juxtaposed bone.Based on the mechanical and cell culture data, CNs in a PU matrix isconsidered for both loading (as a coating material) and non-loadingorthopedic applications while still maintaining the ability to limitfibroblast and promote osteoblast function. Nanophase materials haveincreased numbers of atoms at the surface, greater numbers of materialdefects (such as grain boundaries) at the surface, and increasedelectron delocalization at the surface: properties that will certainlyalter surface energetics of a composite containing nanophase compared toconventional materials. Such altered surface energetics of nanophasematerials have changed interactions of proteins that influencesubsequent cellular adhesion. For example, the conformation of adsorbedvitronectin, a linear protein of 15 nm in length contained in serum thatmediates osteoblast adhesion is different on nanophase compared toconventional grain size ceramics. Compared to conventional ceramics,nanophase ceramics promoted unfolding of vitronectin to exposecell-adhesive epitopes (such as arginine-glycine-aspartic acid, RGD)recognized by specific osteoblast-membrane receptors. Similar alteredprotein interactions important for cell function may be occurring on thenovel composites in the present disclosure.

Interactions of neurons with polymer materials containing carbonnanotubes (CN) were increased. PC-12 cells were seeded onto substratescoated with laminin (5 μg ml⁻¹; Sigma) at 13 500 cells cm⁻² and allowedto differentiate for 3 d. At the end of the time period, the cells wererinsed with PBS, fixed with formaldehyde and stained with DiI (D-282;Molecular Probes). The number of neurites (extensions that may formaxons) were determined per cell after three days as a measure offavorability to the materials of interest. Cell counts were expressed asthe average number of cells in five random fields per square centimeterof substrate surface area. The number of neurites per neuron wasdetermined for each neuron in five random fields per substrate.

First evidence of neurite extension from neurons cultured on 100:0 and98:2 PU:CN wt % formulations are shown in FIG. 2 and FIG. 3. However,there was a slight decrease in the extension of neurites in neuronscultured on composites containing more than 10 wt % CNs and on 100 wt %CNs. Although this decrease was statistically significant (p<0.1), it isimportant to note that, between all substrates, the difference in thenumber of neurites extended per neuron was only on average approximately1.6 (from 5.6 to 4.0 neurites extended per neuron on the 100:0 and 0:100PU:CN wt %, respectively). Since a neurite must extend from a neuronbefore it becomes an axon, these results show the ability of PU:CNformulations to promote interactions with neurons needed for successfulneural probes.

Aligned carbon nanofibers in a polymer matrix is shown in FIG. 13.Random agglomerated carbon nanofibers in the polymer matrix before theapplication of the electric field becomes aligned in the presence of anelectric field. Aligned carbon nanofibers in the polymer matrix in thepresence of an electrical field is observed (FIG. 13). By controllingthe alignment of carbon nanofibers in a polymer matrix, the ability tocontrol axon extension in neurons and control of scar tissue isachieved.

The above indicates that a neural implant coated with or made from asuitable nanomaterial of the present disclosure could be secured in theneurological tissue using known techniques. The nanomaterial in theimplant would also be aligned (as in FIG. 13) to guide axonal growth andreduce scar tissue formation.

Multiwalled carbon nanofibers (CN), synthesized using catalytic andchemical vapor deposition, were acquired from Applied Sciences,Inc./Pyrograf Products, Inc. Composites were formed from 100 nm, lowsurface energy fibers (25-50 mJ/m2) or 60 nm, high surface energy fibers(125-140 mJ/m2) and poly carbonate urethane (Thermedics PolymerProducts, PC3575A). Polycarbonate urethane pellets were allowed todissolve in chloroform at 25° C. for 40 min with sonication, then carbonnanofibers were added and sonication continued for another hour. Thismixture, when viscous, was poured into wells of a microscope slidefashioned with parallel copper plate electrodes. Voltage was applied tothe solution to create a homogeneous electric field. Voltages up to 700were applied to the wells until the composite had cured.

Characterization of surface roughness: Various formulations of carbonnanotubes (CN) at the surface of the polyurethane (PU) matrix werefabricated (from concentrations of 100:0 to 0:100 PU:CN wt %) of addedCNs (FIG. 1). Larger numbers of CNs in the PU matrix increased nanometersurface roughness, and increased elastic modulus (TABLE 1). The elasticmodulus of the 75:25 PU:CN wt % composite was over nine times therespective value measured for pure PU (100 wt %). Although thepercentage elongation was similar for 100:0, 98:2, and 90:10 PU:CN wt %formulations, it was significantly lower for the 75:25 PU:CN wt %. Inaddition, although the tensile strength increased (from 7 to 9 MPa) forthe 98:2 to 90:10 PU:CN wt %, it decreased when the fiber concentrationwas 25 wt % (specifically, at 5 MPa). An optimal concentration ofpolymer to CNs exists where the physical bond between the polymer andthe CNs is at its peak and any increase in CNs from that point mayweaken the material.

Characterization of mechanical and electrical property of nanomaterials:An MTS mechanical testing machine (Sintech) was used to determine thetensile strength, percentage elongation, and elastic modulus of each ofthe substrates (mechanical properties) pertinent to orthopedicapplications. Dry, cut composite strips (40 mm×10 mm) were each testedat a strain rate of 12 mm min⁻¹ using a 10 lb load cell at roomtemperature. Resistance of the nanomaterials was determined usingtetrapolar electrodes with a LRC bridge 2400. The probes were used tomeasure the resistance through a stack of the substrate discs and with asurface area of 1.327 cm² and heights varying from 0.035 to 0.124 cmeach. Resistivity was calculated from the resistance and discmeasurements (TABLE 2).

The mechanical properties of the 100 CN wt % were not measurable. Thetensile strength is about an order of magnitude lower than that ofcortical bone as well as other load bearing implant materials (TABLE 1)while the elastic modulus is almost three orders of magnitude lower. Forthese reasons, the currently designed materials would be best suited asa bone implant coating or in non-loading orthopedic applications,although future studies could perhaps optimize these mechanicalproperties for load bearing applications.

The electrical resistivity values of the substrates are presented inTABLE 2. Greater resistivities were measured for the 98:2 PU:CN wt %composite compared to other formulations containing CNs. The compositewith 10 wt % of CNs exhibited slightly higher resistivity values thanthe 75:25 PU:CN wt % composite. When compared to silicon, which iscommonly used in neural implant applications, the electrical resistivityof the composites disclosed herein, can be altered based upon electricalneeds of an implant such as, for example, a probe. A conductive ornon-conductive part of a neural prosthetic is such an implant that canbe fabricated with desired resistivity levels.

The nanomaterials' effectiveness for reducing the glial scar responsewas tested by way of an adhesion, proliferation, phosphatase activity,and protein content assays with astrocytes, which are described asfollows.

(a) Astrocyte cell culture: Rat astrocyte cells (CRC-11372) werepurchased from the American Type Culture Collection. The cells werecultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco), supplementedwith 10% fetal bovine serum (FBS; Hyclone), and 1%penicillin/streptomycin (P/S; Hyclone) in a standard cell cultureenvironment (37° C., humidified, 5% CO₂/95% air). Passages numbers 32-40were used.

(b) Astrocyte cell adhesion assay: The astrocytes in DMEM (supplementedwith 10% FBS and 1% P/S) were seeded at a density of 3,500 cells/cm²onto the substrates and were cultured in DMEM (supplemented with 10% FBSand 1% P/S) under standard cell culture conditions for one hour. Cellswere then rinsed with phosphate buffered saline (PBS) to removenonadherent cells, fixed with formaldehyde (Fisher Scientific), andstained with Hoescht 33258 dye (Sigma, St. Louis, Mo.). The visible cellnuclei were then counted in five random fields using fluorescencemicroscopy (365 excitation; 400 nm emission).

(c) Astrocyte proliferation assay: The astrocytes in DMEM (supplementedwith 10% FBS and 1% P/S) were seeded at a density of 3,500 cells/cm²onto the substrates and were cultured in DMEM (supplemented with 10% FBSand 1% P/S) under standard cell culture conditions for 1, 3, and 5 days.Media was replaced fresh every other day. At appropriate intervals,cells were rinsed with PBS to remove nonadherent cells, fixed withformaldehyde, and stained with Hoescht 33258 dye. By counting thestained nuclei using fluorescence microscopy (365 excitation; 400 nmemission), and averaging the number of cells in five random fields persquare cm of substrate, cell density was determined.

(d) Protein content assay: The astrocytes (40,000 cells/cm²) were seededonto the substrates and cultured in DMEM (supplemented with 10% FBS and1% P/S) under standard cell culture conditions for 7 and 14 days. Mediawas replaced fresh every other day. At appropriate intervals, the mediawas replaced with distilled water and the cells were lysed during threefreeze-thaw cycles. The total intracellular protein of the lysed cellswas assessed spectrophotometrically using a BCA protein assay kit(Pierce Chemical Co.) and following manufacturer's instructions.Specifically, aliquots of distilled water containing the proteins fromcell lysates were incubated with a solution of copper sulfate andbicinchoninic acid for 30 minutes at 37° C. The absorbance of thesamples was then measured at a light wavelength of 562 nm on aSpectraMax 290 (Molecular Devices, Corp.) with analysis software(SoftMax Pro 3.12; Molecular Devices, Corp.). The protein concentration(expressed in mg) was then determined from a standard curve obtained byrunning albumin concentrations in parallel with the samples.

(e) Phosphatase activity assay: The experimental substrates were seededwith astrocytes at a concentration of 40,000 cells/cm² and were culturedin DMEM (supplemented with 10% FBS and 1% P/S) under standard cellculture conditions for 7 and 14 days. Media was replaced every otherday. Cells were lysed as described in the total intracellular proteincontent section, then the standard method of Lowry was used to resolvealkaline phosphatase activity. Aliquots of the distilled water solutioncontaining cellular protein were incubated with a reaction solutioncontaining 2-amino-2-methyl-1-propanol (pH=10.3) andp-nitrophenylphosphate (Diagnostic Kit #104; Sigma) at 37° C. for 15minutes, then the reaction was terminated with 0.05 N NaOH. Lightabsorbance of the samples was then measured at a wavelength of 410 nm ona SpectraMax 290 (Molecular Devices, Corp.) with analysis software(SoftMax Pro 3.12; Molecular Devices, Corp.). The alkaline phosphataseactivity (expressed as nano-moles of converted p-nitrophenol/min or asSigma units) was then determined from a standard curve obtained byrunning known p-nitrophenol concentrations in parallel with the samples.The alkaline phosphatase activity was normalized by total intracellularprotein and substrate surface area (expressed as Sigma units/mgprotein/square cm).

The surface and other structural properties of nanomaterials weremeasure using scanning electron microscopes. Scanning electronmicrographs (SEM) measurements were used to assess the topography of thesubstrates of interest to the present study. Samples were gold-palladiumsputter-coated at room temperature. All micrographs were taken using aJEOL JSM-840 scanning electron microscope at 5 kV.

Chemical composition of the nanosubstrates was assessed at theUniversity of Washington using electron spectroscopy for chemicalanalysis (ESCA). These analyses were performed on a Surface ScienceInstruments (SSI) X-Probe instrument. A take-off angle of 55° was usedfor acquisitions in the outer 10 nm of the surface. Surface Physicssoftware (Bend, Oreg.) was used to acquire and analyze surfacecomposition data.

Resistance of the nanomaterials was determined using tetrapolarelectrodes with a LRC Bridge 2400. The probes were used to measure theresistance through a stack of the substrate discs and with a surfacearea of 1.327 cm² and heights varying from 0.035 to 0.124 cm each.Resistivity was calculated from the resistance and disc measurements.Data are expressed as mean values+SEM. Statistical analysis wasperformed using ANOVA methods to determine the variance of thequantitative data.

TABLE 1 Mechanical Properties Of Composite Materials Tensile ElasticSubstrate Strength Elongation Modulus (PU:CN wt. %) (MPa) (%) (MPa)100:0   5 450 4 98:2  7 442 6 90:10 9 452 22 75:25 5 27 38   0:100 N/AN/A N/A Cortical Bone [1] 100 10000 Commercially Pure 110000 Titanium[2] Titanium Alloys* [2] 110000-116000 *Values dependent on alloycomposition and processing conditions.

TABLE 2 Electrical Resistivity Substrate Resistivity (PU:CN wt. %)(Ω-cm) 100:0   N/A 98:2  20,500 90:10 625 75:25 0.354   0:100 0.0598Silicon* [34] 10⁻⁴ to 10² *Values dependent on doping and processingconditions.

TABLE 3 Average Composition of Specific Elements on Carbon Fibers ofHigh and Low Surface Energies Type of Carbon Atomic Oxygen Atomic SulfurAtomic Carbon Fiber Percent Percent Percent Conventional With 97.5 ± 0.62.5 ± 0.6 nd Low Surface Energy Conventional With 98.6 ± 0.1 1.0 ± 0.20.4 ± 0.1 High Surface Energy Nanophase With 97.0 ± 0.2 3.0 ± 0.2 nd LowSurface Energy Nanophase With 97.8 ± 0.4 1.7 ± 0.5 0.5 ± 0.1 HighSurface Energy nd = not detected

DOCUMENTS CITED

The following documents are incorporated by reference to the extent theyrelate to protocols used in this disclosure.

-   Hetke, J. F. and Anderson, D. J., “Silicon electrodes for    extracellular recording,” In: Handbook of Neuroprosthetic Methods,    (Finn and LoPresti, eds.), CRC Press LLC, pp. 163-191, 2003).-   Najafi, K (1997) “Micromachined systems for neurophysiological    applications.”, Handbook of microlithography, micromachining, and    microfabrication, Volume II: Micromachining and Microfabrication,    SPIE.-   Neuman et al., (1994) Fabricating Biomedical Sensors with Thin-Film    Technology, IEEE EMB Mag. 13: 409-419.-   Thapa et al., (2003) Nano-structured polymers enhance bladder smooth    muscle cell function Biomaterials 24: 2916-26.-   Wise et al., (1991) Microfabrication Techniques for Integrated    Sensors, and Microsystems. Science 254: 1335-1342.-   U.S. Pat. No. 5,698,175-   U.S. Pat. No. 6,063,243-   U.S. Pat. No. 5,365,073

1-22. (canceled)
 23. An orthopedic prostheses comprising an implantabledevice coated with a composite nanomaterial, said nanomaterialcomprising a polymer matrix and carbon nanotubes having a width of about2 to 200 nm, said device capable of stimulating osteoblast proliferationand minimizing fibroblast encapsulation.
 24. The orthopedic prosthesisof claim 23 wherein the carbon nanotubes have a width of about 10 to 100nm and are functionalized with 4-hydroxynonenal.
 25. The orthopedicprostheses of claim 24 wherein substantially all of the carbon nanotubesare aligned with one another.
 26. The orthopedic prostheses of claim 23wherein said polymer is selected from the group consisting ofpolyurethane, polymethacrylate, polyester, polyvinyl and any copolymersthereof.
 27. The orthopedic prostheses of claim 23 wherein thenanofibers are multi-walled nanotubes.
 28. A method of stimulatingosteoblast proliferation and minimizing fibroblast encapsulation by anorthopedic implant in a patient, said method comprising: (a) obtainingan orthopedic implant comprising a composite nanomaterial, saidnanomaterial comprising a polymer matrix and carbon nanotubes, whereinthe carbon nanotubes have a width of about 10 to 100 nm; and (b)securing the implantable device in said patient where proliferation ofosteoblasts is desired.
 29. The method of claim 28 wherein said carbonnanotubes are functionalized with 4-hydroxynonenal.
 30. The method ofclaim 29 wherein substantially all of the carbon nanotubes are alignedwith one another.
 31. The method of claim 30 wherein said polymer isselected from the group consisting of polyurethane, polymethacrylate,polyester, polyvinyl and any copolymers thereof.
 32. The method of claim31 wherein the nanotubes are multi-walled nanotubes.
 33. The method ofclaim 28 wherein the composite nanomaterial comprises apolyurethane-carbon nanofiber composite, wherein said carbon nanofibershave a width of about 10 to 100 nm and are comprised of carbonnanotubes.
 34. A method of selecting a nanomaterial suitable forimplant, the method comprising: (a) determining structural dimensions ofa biological molecule in a biological tissue; and (b) fabricating thenanomaterial whose surface structural dimension is similar to thebiological molecule.
 35. The method of claim 34, wherein thenanomaterial comprises carbon nanofibers having a width of about 10 to100 nm.
 36. The method of claim 34, wherein the biological molecule islaminin.